Background: A single infusion of AAV5-hFVIII-SQ (valoctocogene roxaparvovec) increased factor VIII (FVIII) levels, reduced annualized bleeding rate, and decreased FVIII utilization in clinical study participants with hemophilia A. Durable expression may be associated with presence of circularized vector DNA episomes in hepatocytes, but this has not been confirmed through liver biopsy in humans.

Aims: To evaluate histopathology and vector DNA form, and distribution in human liver biopsies following AAV-hFVIII-SQ administration.

Methods: Liver biopsies were obtained from 2 participants at week 201 and 140 post infusion with 6×10¹² vg/kg or 4×10¹³ vg/kg AAV5-hFVIII-SQ, respectively. Biopsy samples underwent histopathological examination, in situ hybridization to detect vector genomes, and DNA extraction for molecular analyses. Levels of circular vector genomes that were full-length or contained inverted terminal repeat (ITR) fusions were quantified using drop-phase droplet-digital PCR (ddPCR). Southern blotting was used to visualize circular episome configurations. Liver hFVIII-SQ RNA levels were quantified with reverse transcription followed by ddPCR. All participants provided informed consent. Relevant ethics boards approved the protocol. BioMarin Pharmaceutical provided funding.

Results: Histopathological examination revealed normal liver architecture with mild steatosis and no evidence of steatohepatitis or significant inflammation in either participant. In 6×10¹² vg/kg- and 4×10¹³ vg/kg-treated participants, 1.3% and 32% of hepatocytes, respectively, stained positive for vector genomes in a pan-lobular distribution, similar to previous observations in non-human primates (Figure 1). Circularized full-length vector genomes and ITR-fusions were present as monomers and concatemers in both participants (Figure 2).

Liver hFVIII-SQ RNA levels were 7.67×10² copies/µg (6×10¹² vg/kg-treated participant) and 6.77×10⁴ copies/µg (4×10¹³ vg/kg-treated participant). Analyses of additional participant biopsies will be shared at ISTH.

Conclusions: Mechanisms of AAV gene therapy durability are yet to be determined for hemophilia A. Here, we demonstrate persistent FVIII expression consistent with the presence of circularized, full-length hFVIII-SQ DNA in human liver, nearly 4 years after a single AAV5-hFVIII-SQ infusion.

[Figure 1. In situ hybridization of vector genome DNA (brown) in human liver biopsy samples]

[Figure 2. A) Copies of circularized vector genome per diploid genome, and B) configuration of circular episomes in human biopsy samples]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/first-in-human-liver-biopsy-study-following-gene-therapy-for-hemophilia-a/